Despite a great deal of progress in our understanding of the biochemistry of Ras and its role in tumorigenesis, development of effective therapeutic inhibitors of Ras to date has been disappointing. Given the frequency of Ras mutations in human cancers, there is a critical need to develop targeted inhibitors of this oncoprotein for treatment of patients with Ras-positive tumors. Our proposal represents a novel approach to this challenge. We will develop high affinity reagents that specifically target the four most frequent mutant KRas alleles in human tumors. Aim 1 will utilize the monobody platform as a method to isolate highly specific affinity reagents that target the following KRas mutant proteins: G12D, G12V, G12C, and G13D. The specificity and potency of these reagents will be determined in vitro. Aim 2 will then determine the specificity and selectivity of these KRas mutation specific monobodies at abrogating the tumorigenic phenotype of KRas-driven tumors vs tumors driven by other oncogenes. These studies represent a novel approach toward developing highly specific Ras inhibitory reagents and thus have the potential to make a major impact on cancer therapy. In addition, this project is highly relevant to the NCI's mission of targeting Ras-dependent cancers as evidence by the recent Ras Initiative at the Frederick National Laboratory for Cancer Research.